Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1471-1477
Nested Polymerase Chain Reaction With Sequence-Specific
Primers Typing for HLA-A, -B, and -C Alleles: Detection
of Microchimerism in DR-Matched Individuals
By
Anthony S. Carter,
Lucia Cerundolo,
Mike Bunce,
Dicken D.H. Koo,
Kenneth I. Welsh,
Peter J. Morris, and
Susan V. Fuggle
From the Nuffield Department of Surgery, University of Oxford, John
Radcliffe Hospital, Oxford, UK.
 |
ABSTRACT |
It is widely accepted that donor leukocytes survive within the
recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect
the donor-derived material. The techniques used to detect donor-derived
DNA within recipient peripheral blood rely largely on major
histocompatibility complex class II polymorphism. We and others have
shown that the sensitivity of polymerase chain reaction with
sequence-specific primers (PCR-SSP) typing for HLA class
II alleles can be increased 100-fold by the addition of a primary
amplification step (nested PCR-SSP). We have now extended this
technique to encompass typing for HLA class I alleles, thereby adding
flexibility to microchimerism testing by enabling testing of recipients
HLA-DR matched with their donors. However, the high level of
sensitivity achieved with the technique (1:100,000) leads to a
concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in
the development of our nested PCR-SSP typing system for HLA class II
alleles. We describe here how it is possible to compensate for these
anomalies by including multiple testing of a pretransfusion sample that
acts as a specificity control, establishing a rigorous baseline for
subsequent analysis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IT IS WIDELY ACCEPTED that donor
leukocytes can survive within a recipient after
transplantation1-4 or blood transfusion,5-8 yet
the relevance of this microchimerism remains a contentious issue.4,9-13 Further controlled studies are required to
achieve an improved understanding of the functional importance of microchimerism.
The majority of assays used to detect donor-derived material in
recipient blood exploit sex mismatching between donor and recipient1,14-16 or the polymorphism of the HLA-DR
region of the major histocompatibility complex
(MHC).2,4,9,10,17 The most commonly used techniques based
on HLA-DR polymorphism are the polymerase chain reaction with either
sequence-specific primers (PCR-SSP) or sequence-specific
oligonucleotides (PCR-SSO). These methods are commonly used to HLA type
donors and recipients before transplantation,18,19 but may
be applied to the detection of microchimerism. The sensitivity of
detection of such techniques is variable and can range from 0.5% to
0.01%,3,20,21 but the sensitivity of PCR-SSP
for HLA-DR alleles has been increased by the introduction of a
preliminary PCR (nested PCR-SSP), amplifying exon 2 of
HLA-DRB1.4,17,22,23 It has been demonstrated that the
sensitivity of nested PCR-SSP typing for HLA-DR alleles is at least
100-fold higher than that of standard PCR-SSP typing.17,22 Nevertheless, the use of nested PCR-SSP typing for HLA-DR alleles is
not applicable when the donor and recipient are HLA-DR matched. In such
cases, it would be useful to be able to increase the power of detection
of the technique by applying the recently developed molecular typing
methods for HLA class I alleles.19,24-26
We describe here the development of a nested PCR-SSP typing method for
the detection of HLA class I alleles. During the development of this
method, we detected bands on the gel that were not indicative of donor
or recipient HLA type, entirely consistent with our previous findings
when developing a nested PCR-SSP typing system for HLA-DR alleles.22 These nonspecific bands do not arise from
contamination, but result from the nonspecific amplification of
recipient DNA with certain primer sets. The application of the
technique to clinical samples is described, emphasizing the importance
of rigorous controls for this type of assay system.
| |
MATERIALS AND METHODS |
Clinical material.
Anticoagulated peripheral blood was obtained from selected HLA-typed
healthy volunteers for the sensitivity experiments. To validate the
technique, peripheral blood samples were obtained from patients at the
Oxford Transplant Centre (Oxford, UK) awaiting a renal transplant who
had received planned transfusions of HLA-typed blood. Each patient
received 50 mL of freshly isolated blood (<36 hours from donation)
from 2 healthy donors. Blood samples were obtained at 2 time points
before the transfusion and at defined time points after transfusion.
DNA isolation.
Genomic DNA was isolated from leukocytes obtained from anticoagulated
blood using a salting out procedure,27 was precipitated with ethanol, and was resuspended in sterile water. The genomic DNA was
quantitated and purity was assessed by spectroscopic absorbance at 260 and 280 nm.
Nested PCR-SSP typing.
Two hundred nanograms of DNA was initially amplified in a buffer
containing 67 mmol/L Tris, pH 8.8; 16.6 mmol/L
NH4SO4; 200 µmol/L of each dATP, dCTP, dGTP,
and dTTP; 1.0 mmol/L MgCl2; 0.5 µmol/L forward and
reverse primer (Table 1); and 0.25 U BioTaq polymerase (Bioline, London, UK) for 30 cycles according to Cereb et
al28 in a PTC200-96v thermal cycler (Genetic Research
Instrumentation, Essex, UK). The resultant PCR product was diluted
1:500 in water before PCR-SSP typing. Extreme care was taken to avoid
contamination at any stage.29 Filter tips were used when
pipetting (BioExpress UK Ltd, London, UK), and the work was performed
in a clean air cabinet (Heto-Holten UK Ltd, Camberley, UK).
PCR-SSP typing.
Allele-specific primers (0.5 µmol/L) designed on the basis of
published sequences19,30 (Table
2) were used in multiple amplification
reactions consisting of diluted first-round PCR product (1/500); 67 mmol/L Tris, pH 8.8; 16.6 mmol/L NH4SO4; 200 µmol/L of each dATP, dCTP, dGTP, and dTTP; 2.0 mmol/L
MgCl2; and 0.25 U BioTaq polymerase. PCR amplifications
were performed in a PTC200-96v thermal cycler, and the products were
subsequently analyzed by agarose gel electrophoresis using the
conditions exactly as previously described.19
Sensitivity of nested PCR-SSP typing.
Primer mixes 151 (A*0101-4N), 9 (A*2501-2, A*2601-11N, A*3401-2,
A*6601-3), 155 (B*0801-3), 47 (B*1301-3), 86 (Cw*0102-3), and 106 (Cw*1502-6) (Table 2) were randomly selected to determine the sensitivity of nested PCR typing. The experiments were performed as
previously described and repeated on three occasions.22
Briefly, decreasing amounts of DNA from selected HLA-typed healthy
volunteers were mixed with DNA of known HLA type, both at a starting
concentration of 200 ng/µL, to give final concentrations of 1%,
0.1%, 0.01%, and 0.001% (vol/vol). The mixtures were then subjected
to nested PCR-SSP typing. All products were subsequently analyzed by
agarose gel electrophoresis.
| |
RESULTS |
Sensitivity of nested PCR-SSP typing for HLA class I alleles.
The sensitivity of detection of 6 of the HLA class I primer mixes (151, 9, 155, 47, 86, and 106; see Table 2) was determined by
performing mixing experiments, and the results are shown in Fig 1. Each primer mix was capable of
reproducibly detecting DNA to a level of 0.001% (equivalent to a
dilution of DNA of 1:100,000), demonstrating that the sensitivity of
this technique is comparable to that of other nested PCR-SSP
techniques.17,22
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| Fig 1.
Sensitivity of nested PCR-SSP typing for HLA class I
alleles. (a) Primer mix (PM) 151 (A*0101-4N); (b) PM 9 (A*2501-2,
A*2601-11N, A*3401-2, A*6601-3); (c) PM 155 (B*0801-3); (d) PM 47 (B*1301-3); (e) PM 86 (Cw*0102-3); and (f) PM 106 (Cw*1502-6). DNA of
known HLA type (200 ng/µL) was mixed with an irrelevant DNA (200 ng/µL) to give relative final concentrations of 10% (lane 1), 1%
(lane 2), 0.1% (lane 3), 0.01% (lane 4), and 0.001% (lane 5). Lane 6 is a specificity control in which the irrelevant DNA was amplified on
its own. Each mix was then subject to a primary amplification with the
appropriate set of first-round primers.28 The resultant
product was diluted 1:500 in water before PCR-SSP typing using the
method of Bunce et al.19 The gel is run from
negative ( ) to positive (+). Sensitivity experiments were
performed on 3 occasions, and each primer mix was reproducibly capable
of detecting DNA at a final concentration of 0.001%.
|
|
Application of nested PCR-SSP typing to the detection of
microchimerism.
Samples were investigated from a patient who had received planned
HLA-typed blood transfusions mismatched for HLA class I alleles.
Samples obtained before and 2 days after blood transfusion were
compared (Fig 2). All recipient alleles
were detected in both samples (arrowed) and, in the posttransfusion
samples, additional bands corresponding to the HLA alleles of both
blood transfusion donors were evident (asterisked). However,
nonspecific products also appeared in certain lanes on the gels, a
finding consistent with our results of nested PCR-SSP typing for
HLA-DR.22 The bands were not compatible with the known HLA
type of the donors or the recipient. The pattern of bands generated by
the nested PCR-SSP typing of the pretransfusion sample remains
consistent in the posttransfusion samples and, thus, by inference, is
recipient HLA-type dependent.
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| Fig 2.
Detection of microchimerism using nested PCR-SSP typing.
(A) Pretransfusion DNA sample (recipient HLA type: A*0301,
A*2402; B*4001, B*51011; Cw*0304, Cw*1502, all recipient bands arrowed;
other bands are nonspecific). (B) Posttransfusion DNA sample (blood
donor 1 HLA type: A*0101; B*0801, B*4402; Cw*0701, Cw*0704; blood donor
2 HLA type: A*2501, A*3002; B*3501, B*5501; Cw*0303, Cw*0401).
Asterisked bands are those donor alleles detected; the pattern of
recipient alleles and nonspecific bands is the same as that of a
pretransfusion sample. Patient DNA was isolated from peripheral blood
leukocytes and used in a primary amplification using HLA-A, -B, or -C
primers.28 The resultant products were diluted 1:500 and
used in a PCR-SSP typing system.19 The gel is run from
negative () to positive (+).
|
|
To control for the presence of the nonspecific products resulting from
mispriming, a specificity control in the form of a pretransfusion
sample is required. Rigorous analysis of this sample establishes a
baseline for the analysis of posttransfusion samples. Therefore, nested
PCR-SSP typing is routinely performed 5 times on 2 samples obtained
before transfusion and on samples after transfusion. The results from
one such analysis of the HLA-A locus are shown in
Fig 3.
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| Fig 3.
Validation of nested PCR-SSP typing. Recipient 01 (HLA-A
type: A*0301, A*2402) was transfused with fresh blood from 2 healthy,
HLA-typed volunteers (donor 1: A*0101; donor 2: A*2501, A*3002). The
bar graph shows the results of amplification of 2 pretransfusion
samples and a posttransfusion sample by primer mixes for the HLA-A
locus alleles used in this system (Table 2). Analysis of
both pretransfusion samples produces a similar pattern of amplification
in which primer mixes 4 and 5 represent recipient alleles
(A*0301) and (A*2402), respectively (dark shading). Potentially
informative primer mixes are those that do not give rise to nonspecific
products in any of the multiple tests of the pretransfusion samples. In
this case, these are primer mixes 151, 9, 12, 152, 18, 184, and 154. Analysis of the posttransfusion samples shows additional amplification
with primer mix 151, which is indicative of donor 1 (A*0101), and
primer mixes 9 and 18, which are both indicative of donor 2 (A*2501 and
A*3002, respectively; hatched shading). Primer mixes 3, 6, 10, 13, 14, 15, 153, 23, and 24 detect nonspecific products in the pretransfusion
samples and thus are noninformative in the analysis of posttransfusion
samples (light shading). The requirement for testing pretransfusion
samples is demonstrated by the results with primer mix 10, which
potentially should amplify A*2501 from donor 2. In this example,
although there was amplification in the posttransfusion sample, the
reaction has to be excluded from the analysis because of the
nonspecific amplification present in the pretransfusion
samples.
|
|
The recipient HLA type (A*0301, A*2402) is present in all repeats of
the pretransfusion and posttransfusion samples (PM 4 and 5; dark
shading). Potentially informative reactions are those reactions that do
not yield a band in the pretransfusion samples (in this case, primer
mixes 151, 9, 12, 152, 18, 184, and 154). The blood donors were typed
as HLA-A*0101 (donor 1) and HLA-A*2501, A*3002 (donor 2), and these
alleles are recognized by primer mixes 151 (A*0101), 9 (A*2501), 10 (A*2501), and 18 (A*3002). All of these primer mixes gave rise to a
band when the posttransfusion sample was analyzed, but in the case of
primer mix 10, a band was also evident in both pretransfusion samples.
The reaction with primer mix 10 is therefore termed a noninformative
reaction. Furthermore, there were other primer mixes that gave rise to
bands in the pretransfusion samples that were also present in
posttransfusion samples (PM 3, 6, 13, 14, 15, 153, and 24). The
presence of these nonspecific products is dependent on the recipient
HLA type.
| |
DISCUSSION |
In this report, we have described a method for the detection of HLA
class I donor alleles after blood transfusion using the nested PCR-SSP
technology previously developed for HLA-DR alleles.17,22,31 The technique is finely tuned to achieve maximum sensitivity while retaining specificity; for example, increased sensitivity can be
achieved by increasing the amount of DNA in the first-round amplification, but such a modification will result in a decrease in
specificity. The sensitivity of the technique has been demonstrated using mixing experiments and DNA can be detected to a level of at least
0.001% (equivalent to a dilution of 1:100,000), comparable to the
techniques developed for the detection of HLA-DR
alleles.17,22
The high level of sensitivity achieved with the technique leads to a
concomitant decrease in specificity. When the nested PCR-SSP typing was
applied to the detection of donor-type microchimerism in a patient
receiving an HLA-mismatched blood transfusion, nonspecific products
appeared that did not correspond to the donor or recipient HLA type.
This finding is entirely consistent with our previous results obtained
while developing a similar system for the detection of HLA-DR alleles.
In the previous study, we sequenced several of these nonspecific
products and determined that they did not result from contamination but
rather from mispriming events that led to amplification of
recipient-derived HLA alleles or associated pseudogenes.22
It is therefore possible that some of the nonspecific products seen in
the HLA class I nested PCR-SSP typing system result from the
amplification of HLA class I-associated pseudogenes, of which 6 have
been currently identified.32,33 Therefore, to
use nested PCR-SSP typing to analyze clinical samples, a means of
carefully controlling the system is required.
In the system we have devised, a pretransfusion blood sample is
analyzed to act as a baseline and specificity control. Multiple analysis of this sample with nested PCR-SSP typing allows a rigorous baseline to be established for all subsequent analyses (Figs 2 and 3).
If such a sample is not included in the analysis, then amplification
appearing in posttransfusion samples may be wrongly assumed to be
indicative of donor alleles. For example, in Fig 2, primer mix 10, which amplifies the donor allele HLA-A*2501, yields a band in the
posttransfusion sample suggesting the presence of microchimerism.
However, this band is also detected in the pretransfusion sample;
therefore, the amplification in the posttransfusion samples is
noninformative. The requirement for a pretransfusion/transplant sample
has also been shown by Elwood et al,4 who, by using nested
PCR-SSP typing for HLA-DR alleles, demonstrated that false-positive results for microchimerism would have been obtained in 17% of patients
had a recipient pretransplant DNA sample not been included in the analysis.
The inclusion of HLA-A, -B, and -C alleles in the nested PCR-SSP system
significantly increases the power of this type of analysis where donors
and recipients are frequently matched for HLA-DR alleles. The technique
is flexible and widely applicable to the detection of microchimerism
after blood transfusion or solid organ transplantation and may provide
the means for understanding the true relevance of microchimerism.
| |
FOOTNOTES |
Submitted December 8, 1998; accepted April 20, 1999.
Supported by a grant from the National Kidney Research Fund.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Susan V. Fuggle, D. Phil.,
Nuffield Department of Surgery, University of Oxford, John Radcliffe
Hospital, Oxford, OX3 9DU, UK; e-mail: susan.fuggle{at}nds.ox.ac.uk.
| |
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TOP
ABSTRACT
INTRODUCTION